Gene silencing by the RNA interference (RNAi) machinery is an evolutionary conserved process that is critical for control of genes expression in organisms from yeast to human. Targets of RNAi are recognized through complementary interactions with small RNAs that act as guides in the silencing process. Several discrete classes, encompassing thousands of small RNAs have been identified in recent years. For example, microRNAs interact with members of the ubiquitously expressed Argonaute protein family and together these regulate gene expression networks that impact diverse biological processes. Members ofthe other branch of the Argonaute family, the Piwi proteins, are expressed specifically in germ cells with mutant animals showing severe defects in gametogenesis that lead to sterility. Until recently, the small RNA partners of Piwi proteins were unknown, and the molecular functions of Piwis in gametogenesis remain so. We comprehensively characterized Piwi interacting RNA (piRNA) expression in germ cells of mouse and Drosophila. We have also identified the protein partners of Piwi proteins and additional components ofthe cytoplasmic granules that represent the main sites of piRNA pathway operation. In this proposal I present a focused strategy for analyzing piRNA biogenesis and function in the germline. In aim 1,1 will probe the mechanism of piRNA biogenesis in vivo. In aim 2,1 will determine the mechanism of piRNA-Piwi mediated silencing.